EP2512225A1 - Use of vip3ab in combination with cry1ca for management of resistant insects - Google Patents

Abstract

The subject invention includes methods and plants for controlling fall army worm lepidopteran insects, said plants comprising a V1p3Ab insecticidal protein and a Cry1Ca insecticidal protein, and various combinations of other proteins comprising this pair of proteins, to delay or prevent development of resistance by the insects.

Description

USE OF Vip3Ab IN COMBINATION WITH CrylCa FOR MANAGEMENT OF RESISTANT INSECTS

Background of the Invention

[0001] Humans grow corn for food and energy applications. Humans also grow many other crops, including soybeans and cotton. Insects eat and damage plants and thereby undermine these human efforts. Billions of dollars are spent each year to control insect pests and additional billions are lost to the damage they inflict. Synthetic organic chemical insecticides have been the primary tools used to control insect pests but biological insecticides, such as the insecticidal proteins derived from Bacillus thuringiensis (Bt), have played an important role in some areas. The ability to produce insect-resistant plants through transformation with Bt insecticidal protein genes has revolutionized modern agriculture and heightened the importance and value of insecticidal proteins and their genes.

[0002] Several Bt proteins have been used to create the insect-resistant transgenic plants that have been successfully registered and commercialized to date. These include CrylAb, CrylAc, CrylF and Cry3Bb in corn, CrylAc and Cry2Ab in cotton, and Cry3A in potato.

[0003] The commercial products expressing these proteins express a single protein except in cases where the combined insecticidal spectrum of 2 proteins is desired (e.g., CrylAb and Cry3Bb in corn combined to provide resistance to lepidopteran pests and rootworm, respectively) or where the independent action of the proteins makes them useful as a tool for delaying the development of resistance in susceptible insect populations (e.g., CrylAc and Cry2Ab in cotton combined to provide resistance management for tobacco budworm). See also U.S. Patent Application Publication No. 2009/0313717, which relates to a Cry2 protein plus a Vip3Aa, CrylF, or CrylA for control of Helicoverpa zea or armigerain. WO 2009/132850 relates to CrylF or CrylA and Vip3Aa for controlling Spodoptera frugiperda. U.S. Patent Application Publication No. 2008/031 1096 relates in part to CrylAb for controlling CrylF-resistant ECB.

[0004] That is, some of the qualities of insect-resistant transgenic plants that have led to rapid and widespread adoption of this technology also give rise to the concern that pest populations will develop resistance to the insecticidal proteins produced by these plants. Several strategies have been suggested for preserving the utility of 5?-based insect resistance traits which include deploying proteins at a high dose in combination with a refuge, and alternation with, or co-deployment of, different toxins (McGaughey et al.

(1998), "5.?. Resistance Management," Nature Biotechnol. 16: 144-146).

[0005] The proteins selected for use in an insect resistant management (IRM) stack need to exert their insecticidal effect independently so that resistance developed to one protein does not confer resistance to the second protein (i.e., there is not cross resistance to the proteins). If, for example, a pest population that is resistant to "Protein A" is sensitive to "Protein B", one would conclude that there is not cross resistance and that a combination of Protein A and Protein B would be effective in delaying resistance to Protein A alone.

[0006] In the absence of resistant insect populations, assessments can be made based on other characteristics presumed to be related to mechanism of action and cross-resistance potential. The utility of receptor-mediated binding in identifying insecticidal proteins likely to not exhibit cross resistance has been suggested (van Mellaert et al. 1999). The key predictor of lack of cross resistance inherent in this approach is that the insecticidal proteins do not compete for receptors in a sensitive insect species.

[0007] In the event that two Bt toxins compete for the same receptor, then if that receptor mutates in that insect so that one of the toxins no longer binds to that receptor and thus is no longer insecticidal against the insect, it might be the case that the insect will also be resistant to the second toxin (which competitively bound to the same receptor). That is, the insect is said to be cross-resistant to both Bt toxins. However, if two toxins bind to two different receptors, this could be an indication that the insect would not be simultaneously resistant to those two toxins.

[0008] For example, CrylFa protein is useful in controlling many lepidopteran pests species including the European corn borer (ECB; Ostrinia nubilalis (Hubner)) and the fall armyworm (FAW; Spodoptera frugiperda), and is active against the sugarcane borer (SCB; Diatraea saccharalis) . The CrylFa protein, as produced in transgenic corn plants containing event TCI 507, is responsible for an industry-leading insect resistance trait for FAW control. CrylFa is further deployed in the Herculex^®, Smarts tax™, and

WideStrike™ products.

[0009] Additional Cry toxins are listed at the website of the official B. t. nomenclature committee (Crickmore et al.; lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/). There are currently nearly 60 main groups of "Cry" toxins (Cryl-Cry59), with additional Cyt toxins and VIP toxins and the like. Many of each numeric group have capital-letter subgroups, and the capital letter subgroups have lower-cased letter sub-subgroups. (Cryl has A-L, and CrylA has a-i, for example).

Brief Summary of the Invention

[0010] The subject invention relates in part to the use of a Vip3Ab protein in combination with a CrylCa protein. Plants (and acreage planted with such plants) that produce both of these proteins are included within the scope of the subject invention.

[0011] The subject invention relates in part to the surprising discovery that Vip3Ab does not compete with CrylCa for binding sites in the gut of fall army worm (Spodoptera frugiperda; FAW).

[0012] The subject invention also relates in part to triple stacks or "pyramids" of three (or more) toxins, with Vip3Ab and CrylCa being the base pair. In some preferred pyramid embodiments, the combination of the selected toxins provides non-cross-resistant action against FAW. Some preferred "three sites of action" pyramid combinations include the subject base pair of proteins plus CrylFa, CrylDa, CrylBe, or CrylE as the third protein for targeting FAW. These particular triple stacks would, according to the subject invention, advantageously and surprisingly provide three sites of action against FAW. This can help to reduce or eliminate the requirement for refuge acreage.

[0013] Additional toxins/genes can also be added according to the subject invention. For example, if CrylFa or CrylBe are stacked with the subject pair of proteins (both CrylFa and CrylBe are both active against both FAW and European cornborer (ECB)), adding two additional proteins to this triple stack wherein the two added proteins target ECB, would provide three sites of action against FAW, and three sites of action against ECB. These two added proteins (the fourth and fifth proteins) could be selected from the group consisting of Cry2A, Cryll, DIG-3, and CrylAb. This would result in a five-protein stack having three sites of action against two insects (ECB and FAW).

DETAILED DESCRIPTION OF THE INVENTION

[0014] The subject invention relates in part to the surprising discovery that Vip3Ab and CrylCa do not compete for binding with each other in the gut of fall army worms (FAW; Spodoptera frugiperda). Thus, a Vip3Ab protein can be used in combination with a CrylCa protein in transgenic corn (and other plants; e.g., cotton and soybeans, for example) to delay or prevent FAW from developing resistance to either of these proteins alone. The subject pair of proteins can be effective at protecting plants (such as maize plants and/or soybean plants) from damage by Cry-resistant fall army worm. That is, one use of the subject invention is to protect corn and other economically important plant species from damage and yield loss caused by fall armyworm populations that could develop resistance to Vip3Ab or CrylCa.

[0015] The subject invention thus teaches an insect resistant management (IRM) stack comprising Vip3Ab and CrylCa to prevent or mitigate the development of resistance by FAW to either or both of these proteins.

[0016] The present invention provides compositions for controlling lepidopteran pests comprising cells that produce a Vip3Ab insecticidal protein and a CrylCa insecticidal protein.

[0017] The invention further comprises a host transformed to produce both a Vip3Ab insecticidal protein and a CrylCa insecticidal protein, wherein said host is a microorganism or a plant cell. The subject polynucleotide(s) are preferably in a genetic construct under control of a non-Bacillus-thuringiensis promoter(s). The subject polynucleotides can comprise codon usage for enhanced expression in a plant.

[0018] It is additionally intended that the invention provides a method of controlling lepidopteran pests comprising contacting said pests or the environment of said pests with an effective amount of a composition that contains a Vip3 Ab core toxin-containing protein and further contains a CrylCa core toxin-containing protein.

[0019] An embodiment of the invention comprises a maize plant comprising a plant- expressible gene encoding a CrylCa insecticidal protein and a plant-expressible gene encoding a Vip3 Ab insecticidal protein, and seed of such a plant.

[0020] A further embodiment of the invention comprises a maize plant wherein a plant- expressible gene encoding a CrylCa insecticidal protein and a plant-expressible gene encoding a Vip3Ab insecticidal protein have been introgressed into said maize plant, and seed of such a plant.

[0021] As described in the Examples, competitive receptor binding studies using radiolabeled CrylCa protein show that the CrylCa protein does not compete for binding in FAW tissues to which Vip3Ab binds. These results also indicate that the combination of Vip3Ab and CrylCa proteins can be an effective means to mitigate the development of resistance in FAW populations to either of these proteins. Thus, based in part on the data described herein, it is thought that co-production (stacking) of the CrylCa and Vip3Ab proteins can be used to produce a high dose IRM stack for FAW.

[0022] Other proteins can be added to this pair. For example, the subject invention also relates in part to triple stacks or "pyramids" of three (or more) toxins, with Vip3Ab and CrylCa being the base pair. In some preferred pyramid embodiments, the selected toxins have three separate sites of action against FAW. Some preferred "three sites of action" pyramid combinations include the subject base pair of proteins plus CrylFa, CrylDa, Cry 1 Be, or Cry IE as the third protein for targetting FAW. By "separate sites of action," it is meant any of the given proteins do not cause cross-resistance with each other. These particular triple stacks would, according to the subject invention, advantageously and surprisingly provide three sites of action against FAW. This can help to reduce or eliminate the requirement for refuge acreage.

[0023] Related to some specific embodiments of the subject invention, we showed that a FAW population resistant to the insecticidal activity of the CrylFa protein is not resistant to the insecticidal activity of the Vip3Ab protein or to the insecticidal activity of the CrylCa protein. We demonstrated that CrylCa does not compete for the binding sites with CrylFa and that Vip3Ab does not compete for the binding sites with CrylFa in the gut of FAW. See USSN 61/284,281 (filed December 16, 2009) regarding CrylFa and CrylCa, and concurrently filed PCT application entitled "COMBINED USE OF Vip3Ab AND CRY 1 Fa FOR MANAGEMENT OF RESISTANT INSECTS ")

[0024] Thus, the subject pairs of toxins CrylFa plus Vip3Ab and CrylFa plus CrylCa provide non-cross-resistant action against FAW. The inability of Vip3Abl to compete for the binding of CrylCa in the gut of FAW demonstrates that these three protein toxins (CrylFa, Vip3Ab, and CrylCa) represent a triple-stack pyramid of Cry toxins that provide three separate target site interactions within the gut of FAW. These particular triple stacks would, according to the subject invention, advantageously and surprisingly provide non- cross-resistant action against FAW. Furthermore, by the demonstration that these three proteins do not compete with each other, one skilled in the art will recognize that this can help to reduce or eliminate the requirement for refuge acreage. As with the benefit of this disclosure, plants expressing the triple combination of CrylFa, Vip3Ab and CrylCa, will be useful in delaying or preventing the development of resistance in FAW to the individual or combination of these proteins. [0025] Additional toxins/genes can also be added according to the subject invention. For example, if CrylFa or CrylBe are stacked with the subject pair of proteins (both CrylFa and CrylBe are both active against both FAW and European cornborer (ECB)), adding two additional proteins to this triple stack wherein the two added proteins target ECB, would provide three sites of action against FAW, and three sites of action against ECB. These two added proteins (the fourth and fifth proteins) could be selected from the group consisting of Cry2A, Cry II, DIG-3 (see U.S. Patent Application Serial No. 61/284,278 (filed December 16, 2009) and US 2010 00269223), and CrylAb. This would result in a five-protein stack having three sites of action against two insects (ECB and FAW)

[0026] Thus, one deployment option is to use the subject pair of proteins in combination with a third toxin/gene, and to use this triple stack to mitigate the development of resistance in FAW to any of these toxins. Accordingly, the subject invention also relates in part to triple stacks or "pyramids" of three (or more) toxins. In some preferred pyramid embodiments, the selected toxins have three separate sites of action against FAW.

[0027] Included among deployment options of the subject invention would be to use two, three, or more proteins of the subject proteins in crop-growing regions where FAW can develop resistant populations.

[0028] With CrylFa being active against FAW and ECB, Vip3Ab plus CrylCa plus CrylFa would, according to the subject invention, advantageously and surprisingly provide three sites of action against FAW. This can help to reduce or eliminate the requirement for refuge acreage.

[0029] CrylFa is deployed in the Herculex^®, SmartStax™, and Wides Strike™ products. The subject pair of genes (Vip3Ab and CrylCa) could be combined into, for example, a CrylFa product such as Herculex^®, SmartStax™, and WideStrike™. Accordingly, the subject pair of proteins could be significant in reducing the selection pressure on these andother proteins. The subject pair of proteins could thus be used as in the three gene combinations for corn and other plants (cotton and soybeans, for example).

[0030] As discussed above, additional toxins/genes can also be added according to the subject invention. For the use of Cry IE (for controlling FAW), see U.S. Patent Application Serial No. 61/284,278 (filed December 16, 2009).

[0031] Plants (and acreage planted with such plants) that produce any of the subject combinations of proteins are included within the scope of the subject invention. Additional toxins/genes can also be added, but the particular stacks discussed above advantageously and surprisingly provide multiple sites of action against FAW and/or ECB. This can help to reduce or eliminate the requirement for refuge acreage. A field thus planted of over ten acres is thus included within the subject invention.

[0032] GENBANK can also be used to obtain the sequences for any of the genes and proteins disclosed or mentioned herein. See Appendix A, below. Relevant sequences are also available in patents. For example, U.S. Patent No. 5,188,960 and U.S. Patent No. 5,827,514 describe CrylFa core toxin containing proteins suitable for use in carrying out the present invention. U.S. Patent No. 6,218,188 describes plant-optimized DNA sequences encoding CrylFa core toxin-containing proteins that are suitable for use in the present invention. USSN 61/284,275 (filed December 16, 2009) provides some truncated Cryl Ca proteins that can be used according to the subject invention.

[0033] Combinations of proteins described herein can be used to control lepidopteran pests. Adult lepidopterans, for example, butterflies and moths, primarily feed on flower nectar and are a significant effector of pollination. Nearly all lepidopteran larvae, i.e., caterpillars, feed on plants, and many are serious pests. Caterpillars feed on or inside foliage or on the roots or stem of a plant, depriving the plant of nutrients and often destroying the plant's physical support structure. Additionally, caterpillars feed on fruit, fabrics, and stored grains and flours, ruining these products for sale or severely diminishing their value. As used herein, reference to lepidopteran pests refers to various life stages of the pest, including larval stages.

[0034J Some chimeric toxins of the subject invention comprise a full N-terminal core toxin portion of a Bt toxin and, at some point past the end of the core toxin portion, the protein has a transition to a heterologous protoxin sequence. The N-terminal, insecticidally active, toxin portion of a Bt toxin is referred to as the "core" toxin. The transition from the core toxin segment to the heterologous protoxin segment can occur at approximately the toxin/protoxin junction or, in the alternative, a portion of the native protoxin (extending past the core toxin portion) can be retained, with the transition to the heterologous protoxin portion occurring downstream.

[0035?] As an example, one chimeric toxin of the subject invention, is a full core toxin portion of CrylCa (roughly the first 600 amino acids) and/or a heterologous protoxin (the remaining amino acids to the C-terminus). In one preferred embodiment, the portion of a chimeric toxin comprising the protoxin is derived from a CrylAb protein toxin. In a preferred embodiment, the portion of a chimeric toxin comprising the protoxin is derived from a CrylAb protein toxin.

[0036] A person skilled in this art will appreciate that Bt toxins, even within a certain class such as CrylCa, will vary to some extent in length and the precise location of the transition from core toxin portion to protoxin portion. Typically, the CrylCa toxins are about 1150 to about 1200 amino acids in length. The transition from core toxin portion to protoxin portion will typically occur at between about 50% to about 60% of the full length toxin. The chimeric toxin of the subject invention will include the full expanse of this N-terminal core toxin portion. Thus, the chimeric toxin will comprise at least about 50% of the full length of the Cryl Bt toxin protein. This will typically be at least about 590 amino acids. With regard to the protoxin portion, the full expanse of the CrylAb protoxin portion extends from the end of the core toxin portion to the C-terminus of the molecule.

[0037] Genes and toxins. The genes and toxins useful according to the subject invention include not only the full length sequences disclosed but also fragments of these sequences, variants, mutants, and fusion proteins which retain the characteristic pesticidal activity of the toxins specifically exemplified herein. As used herein, the terms "variants" or

"variations" of genes refer to nucleotide sequences which encode the same toxins or which encode equivalent toxins having pesticidal activity. As used herein, the term "equivalent toxins" refers to toxins having the same or essentially the same biological activity against the target pests as the claimed toxins.

[0038] As used herein, the boundaries represent approximately 95% (Vip3Ab 's and

CrylCa's), 78% (Vip3Ab 's and CrylC's), and 45% (Cryl 's) sequence identity, per

"Revision of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal Proteins," N. Crickmore, D.R. Zeigler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, and D.H. Dean. Microbiology and Molecular Biology Reviews (1998) Vol 62: 807-813. These cut offs can also be applied to the core toxins only.

[0039] It should be apparent to a person skilled in this art that genes encoding active toxins can be identified and obtained through several means. The specific genes or gene portions exemplified herein may be obtained from the isolates deposited at a culture depository. These genes, or portions or variants thereof, may also be constructed synthetically, for example, by use of a gene synthesizer. Variations of genes may be readily constructed using standard techniques for making point mutations. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Genes that encode active fragments may also be obtained using a variety of restriction enzymes. Proteases may be used to directly obtain active fragments of these protein toxins.

[0040] Fragments and equivalents which retain the pesticidal activity of the exemplified toxins would be within the scope of the subject invention. Also, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequences disclosed herein. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same, or essentially the same, toxins. These variant DNA sequences are within the scope of the subject invention. As used herein, reference to "essentially the same" sequence refers to sequences which have amino acid substitutions, deletions, additions, or insertions which do not materially affect pesticidal activity. Fragments of genes encoding proteins that retain pesticidal activity are also included in this definition.

[0041] A further method for identifying the genes encoding the toxins and gene portions useful according to the subject invention is through the use of oligonucleotide probes. These probes are detectable nucleotide sequences. These sequences may be detectable by virtue of an appropriate label or may be made inherently fluorescent as described in International Application No. WO93/16094. As is well known in the art, if the probe molecule and nucleic acid sample hybridize by forming a strong bond between the two molecules, it can be reasonably assumed that the probe and sample have substantial homology. Preferably, hybridization is conducted under stringent conditions by techniques well-known in the art, as described, for example, in Keller, G. FL, M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., pp. 169-170. Some examples of salt concentrations and temperature combinations are as follows (in order of increasing stringency): 2X SSPE or SSC at room temperature; IX SSPE or SSC at 42° C; 0.1X SSPE or SSC at 42° C; 0. IX SSPE or SSC at 65° C. Detection of the probe provides a means for determining in a known manner whether hybridization has occurred. Such a probe analysis provides a rapid method for identifying toxin-encoding genes of the subject invention. The nucleotide segments which are used as probes according to the invention can be synthesized using a DNA synthesizer and standard procedures. These nucleotide sequences can also be used as PCR primers to amplify genes of the subject invention. [0042] Variant toxins. Certain toxins of the subject invention have been specifically exemplified herein. Since these toxins are merely exemplary of the toxins of the subject invention, it should be readily apparent that the subject invention comprises variant or equivalent toxins (and nucleotide sequences coding for equivalent toxins) having the same or similar pesticidal activity of the exemplified toxin. Equivalent toxins will have amino acid homology with an exemplified toxin. This amino acid homology will typically be greater than 75%, preferably be greater than 90%, and most preferably be greater than 95%. The amino acid homology will be highest in critical regions of the toxin which account for biological activity or are involved in the determination of three-dimensional configuration which ultimately is responsible for the biological activity. In this regard, certain amino acid substitutions are acceptable and can be expected if these substitutions are in regions which are not critical to activity or are conservative amino acid substitutions which do not affect the three-dimensional configuration of the molecule. For example, amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. Below is a listing of examples of amino acids belonging to each class.

[0043] In some instances, non-conservative substitutions can also be made. The critical factor is that these substitutions must not significantly detract from the biological activity of the toxin.

[0044] Recombinant hosts. The genes encoding the toxins of the subject invention can be introduced into a wide variety of microbial or plant hosts. Expression of the toxin gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. Conjugal transfer and recombinant transfer can be used to create a Bt strain that expresses both toxins of the subject invention. Other host organisms may also be transformed with one or both of the toxin genes then used to accomplish the synergistic effect. With suitable microbial hosts, e.g., Pseudomonas, the microbes can be applied to the situs of the pest, where they will proliferate and be ingested. The result is control of the pest. Alternatively, the microbe hosting the toxin gene can be treated under conditions that prolong the activity of the toxin and stabilize the cell. The treated cell, which retains the toxic activity, then can be applied to the environment of the target pest.

[0045] Where the Bt toxin gene is introduced via a suitable vector into a microbial host, and said host is applied to the environment in a living state, it is essential that certain host microbes be used. Microorganism hosts are selected which are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops of interest. These microorganisms are selected so as to be capable of successfully competing in the particular environment (crop and other insect habitats) with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.

[0046] A large number of microorganisms are known to inhabit the phylloplane (the surface of the plant leaves) and/or the rhizosphere (the soil surrounding plant roots) of a wide variety of important crops. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., genera Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas,

Methylophilius, Agrobactenum, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae,

Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobactenium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms.

[0047] A wide variety of methods is available for introducing a Bt gene encoding a toxin into a microorganism host under conditions which allow for stable maintenance and expression of the gene. These methods are well known to those skilled in the art and are described, for example, in U.S. Patent No. 5, 135,867, which is incorporated herein by reference.

[0048] Treatment of cells. Bacillus thuringiensis or recombinant cells expressing the Bt toxins can be treated to prolong the toxin activity and stabilize the cell. The pesticide microcapsule that is formed comprises the Bt toxin or toxins within a cellular structure that has been stabilized and will protect the toxin when the microcapsule is applied to the environment of the target pest. Suitable host cells may include either prokaryotes or eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms which produce substances toxic to higher organisms could be used, where the toxic substances are unstable or the level of application sufficiently low as to avoid any possibility of toxicity to a mammalian host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi.

[0049] The cell will usually be intact and be substantially in the proliferative form when treated, rather than in a spore form, although in some instances spores may be employed.

[0050] Treatment of the microbial cell, e.g., a microbe containing the B.t. toxin gene or genes, can be by chemical or physical means, or by a combination of chemical and/or physical means, so long as the technique does not deleteriously affect the properties of the toxin, nor diminish the cellular capability of protecting the toxin. Examples of chemical reagents are halogenating agents, particularly halogens of atomic no. 17-80. More particularly, iodine can be used under mild conditions and for sufficient time to achieve the desired results. Other suitable techniques include treatment with aldehydes, such as glutaraldehyde; anti-infectives, such as zephiran chloride and cetylpyridinium chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Lugol iodine, Bouin's fixative, various acids and Helly's fixative (See: Humason, Gretchen L., Animal Tissue Techniques, W. H. Freeman and Company, 1967); or a combination of physical (heat) and chemical agents that preserve and prolong the activity of the toxin produced in the cell when the cell is administered to the host environment. Examples of physical means are short wavelength radiation such as gamma-radiation and X-radiation, freezing, UV irradiation, lyophilization, and the like. Methods for treatment of microbial cells are disclosed in U.S. Pat. Nos. 4,695,455 and 4,695,462, which are incorporated herein by reference. [0051] The cells generally will have enhanced structural stability which will enhance resistance to environmental conditions. Where the pesticide is in a proform, the method of cell treatment should be selected so as not to inhibit processing of the proform to the mature form of the pesticide by the target pest pathogen. For example, formaldehyde will crosslink proteins and could inhibit processing of the proform of a polypeptide pesticide. The method of treatment should retain at least a substantial portion of the bio-availability or bioactivity of the toxin.

[0052] Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the B.t. gene or genes into the host, availability of expression systems, efficiency of expression, stability of the pesticide in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; survival in aqueous environments; lack of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.

[0053] Growth of cells. The cellular host containing the B.t. insecticidal gene or genes may be grown in any convenient nutrient medium, where the DNA construct provides a selective advantage, providing for a selective medium so that substantially all or all of the cells retain the B.t. gene. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.

[0054] The B.t. cells producing the toxins of the invention can be cultured using standard art media and fermentation techniques. Upon completion of the fermentation cycle the bacteria can be harvested by first separating the B.t. spores and crystals from the fermentation broth by means well known in the art. The recovered B.t. spores and crystals can be formulated into a wettable powder, liquid concentrate, granules or other formulations by the addition of surfactants, dispersants, inert carriers, and other components to facilitate handling and application for particular target pests. These formulations and application procedures are all well known in the art.

[0055] Formulations. Formulated bait granules containing an attractant and spores, crystals, and toxins of the B.t. isolates, or recombinant microbes comprising the genes obtainable from the B.t. isolates disclosed herein, can be applied to the soil. Formulated product can also be applied as a seed-coating or root treatment or total plant treatment at later stages of the crop cycle. Plant and soil treatments of B.t. cells may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, or the like. The ingredients may include rheological agents, surfactants, emulsifiers, dispersants, or polymers.

[0056] As would be appreciated by a person skilled in the art, the pesticidal concentration will vary widely depending upon the nature of the particular formulation, particularly whether it is a concentrate or to be used directly. The pesticide will be present in at least 1% by weight and may be 100% by weight. The dry formulations will have from about 1-95% by weight of the pesticide while the liquid formulations will generally be from about 1 -60% by weight of the solids in the liquid phase. The formulations will generally have from about 10² to about 10⁴ cells/mg. These formulations will be administered at about 50 mg (liquid or dry) to 1 kg or more per hectare.

[0057] The formulations can be applied to the environment of the lepidopteran pest, e.g., foliage or soil, by spraying, dusting, sprinkling, or the like.

[0058] Plant transformation. A preferred recombinant host for production of the insecticidal proteins of the subject invention is a transformed plant. Genes encoding Bt toxin proteins, as disclosed herein, can be inserted into plant cells using a variety of techniques which are well known in the art. For example, a large number of cloning vectors comprising a replication system in Escherichia coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants. The vectors comprise, for example, pBR322, pUC series, M13mp series, pACYC184, inter alia. Accordingly, the DNA fragment having the sequence encoding the Bt toxin protein can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli. The E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each plasmid sequence can be cloned in the same or other plasmids. Depending on the method of inserting desired genes into the plant, other DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, has to be joined as the flanking region of the genes to be inserted. The use of T-DNA for the transformation of plant cells has been intensively researched and sufficiently described in EP 120 516, Lee and Gelvin (2008), Hoekema (1985), Fraley et ah, (1986), and An et ah, (1985), and is well established in the art.

[0059] Once the inserted DNA has been integrated in the plant genome, it is relatively stable. The transformation vector normally contains a selectable marker that confers on the transformed plant cells resistance to a biocide or an antibiotic, such as Bialaphos,

Kanamycin, G418, Bleomycin, or Hygromycin, inter alia. The individually employed marker should accordingly permit the selection of transformed cells rather than cells that do not contain the inserted DNA.

[0060] A large number of techniques are available for inserting DNA into a plant host cell. Those techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, fusion, injection, biolistics

(microparticle bombardment), or electroporation as well as other possible methods. If Agrobacteria are used for the transformation, the DNA to be inserted has to be cloned into special plasmids, namely either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid by homologous

recombination owing to sequences that are homologous to sequences in the T-DNA. The Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA.

Intermediate vectors cannot replicate themselves in Agrobacteria. The intermediate vector can be transferred into Agrobacterium tumefaciens by means of a helper plasmid

(conjugation). Binary vectors can replicate themselves both in E. coli and in Agrobacteria. They comprise a selection marker gene and a linker or polylinker which are framed by the Right and Left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters et ah, 1978). The Agrobacterium used as host cell is to comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained. The bacterium so transformed is used for the transformation of plant cells. Plant explants can advantageously be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the plant cell. Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension- cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection. The plants so obtained can then be tested for the presence of the inserted DNA. No special demands are made of the plasmids in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives.

[0061] The transformed cells grow inside the plants in the usual manner. They can form germ cells and transmit the transformed trait(s) to progeny plants. Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties.

[0062] In a preferred embodiment of the subject invention, plants will be transformed with genes wherein the codon usage has been optimized for plants. See, for example, U.S. Patent No. 5,380,831, which is hereby incorporated by reference. While some truncated toxins are exemplified herein, it is well-known in the Bt art that 130 kDa-type (full-length) toxins have an N-terminal half that is the core toxin, and a C-terminal half that is the protoxin "tail." Thus, appropriate "tails" can be used with truncated / core toxins of the subject invention. See e.g. U.S. Patent No. 6,218,188 and U.S. Patent No. 6,673,990. In addition, methods for creating synthetic Bt genes for use in plants are known in the art (Stewart and Burgin, 2007). One non-limiting example of a preferred transformed plant is a fertile maize plant comprising a plant expressible gene encoding a Vip3Ab protein, and further comprising a second plant expressible gene encoding a CrylCa protein.

[0063] Transfer (or introgression) of the Vip3Ab - and CrylCa-determined trait(s) into inbred maize lines can be achieved by recurrent selection breeding, for example by backcrossing. In this case, a desired recurrent parent is first crossed to a donor inbred (the non-recurrent parent) that carries the appropriate gene(s) for the Vip3Ab - and Cry 1 (redetermined traits. The progeny of this cross is then mated back to the recurrent parent followed by selection in the resultant progeny for the desired trait(s) to be transferred from the non-recurrent parent. After three, preferably four, more preferably five or more generations of backcrosses with the recurrent parent with selection for the desired trait(s), the progeny will be heterozygous for loci controlling the trait(s) being transferred, but will be like the recurrent parent for most or almost all other genes (see, for example, Poehlman & Sleper (1995) Breeding Field Crops, 4th Ed., 172-175; Fehr (1987) Principles of Cultivar Development, Vol. 1 : Theory and Technique, 360-376).

[0064] Insect Resistance Management (IRM) Strategies. Roush et al., for example, outlines two-toxin strategies, also called "pyramiding" or "stacking," for management of insecticidal transgenic crops. (The Royal Society. Phil. Trans. R. Soc. Lond. B. (1998) 353, 1777- 1786).

[0065] On their website, the United States Environmental Protection Agency

(epa.gov/oppbppdl/biopesticides/pips/bt_corn_refuge_2006.htm) publishes the following requirements for providing non-transgenic (i.e., non-B.t.) refuges (a section of non-Bt crops

/ corn) for use with transgenic crops producing a single Bt protein active against target pests.

"The specific structured requirements for corn borer-protected Bt (CrylAb or Cry IF) corn products are as follows:

Structured refuges: 20% non-Lepidopteran Bt corn refuge in Corn Belt;

50% non-Lepidopteran Bt refuge in Cotton Belt

Blocks

Internal (i.e., within the Bt field)

External (i.e., separate fields within ½ mile (¼ mile if possible) of the

Bt field to maximize random mating)

In-field Strips

Strips must be at least 4 rows wide (preferably 6 rows) to reduce

the effects of larval movement"

[0066] In addition, the National Corn Growers Association, on their website:

(ncga.com/insect-resistance-management-fact-sheet-bt-corn)

[0067] also provides similar guidance regarding the refuge requirements. For example:

"Requirements of the Corn Borer IRM:

-Plant at least 20% of your corn acres to refuge hybrids

-In cotton producing regions, refuge must be 50%>

-Must be planted within 1/2 mile of the refuge hybrids

-Refuge can be planted as strips within the Bt field; the refuge strips must be at least 4 rows wide

-Refuge may be treated with conventional pesticides only if economic thresholds are reached for target insect

-Bt-based sprayable insecticides cannot be used on the refuge corn

-Appropriate refuge must be planted on every farm with Bt corn"

[0068] As stated by Roush et al. (on pages 1780 and 1784 right column, for example), stacking or pyramiding of two different proteins each effective against the target pests and with little or no cross-resistance can allow for use of a smaller refuge. Roush suggests that for a successful stack, a refuge size of less than 10% refuge, can provide comparable resistance management to about 50% refuge for a single (non-pyramided) trait. For currently available pyramided Bt corn products, the U.S. Environmental Protection Agency requires significantly less (generally 5%) structured refuge of non-Bt corn be planted than for single trait products (generally 20%).

[0069] There are various ways of providing the IRM effects of a refuge, including various geometric planting patterns in the fields (as mentioned above) and in-bag seed mixtures, as discussed further by Roush et al. (supra), and U.S. Patent No. 6,551,962.

[0070] The above percentages, or similar refuge ratios, can be used for the subject double or triple stacks or pyramids. For triple stacks with three sites of action against a single target pest, a goal would be zero refuge (or less than 5% refuge, for example). This is particularly true for commercial acreage - of over 10 acres for example.

[0071] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety to the extent they are not inconsistent with the explicit teachings of this specification.

[0072] Unless specifically indicated or implied, the terms "a", "an", and "the" signify "at least one" as used herein.

[0073] Following are examples that illustrate procedures for practicing the invention.

These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. All temperatures are in degrees Celsius.

EXAMPLES

Example 1- Production and trypsin processing of Vip3Ab and CrylCa proteins.

The genes encoding the CrylCa and Vip3Abl pro toxins were expressed in Pseudomonas fluorescens expression strains and the full length proteins isolated as insoluble inclusion bodies. The washed inclusion bodies were solubilized by stirring at 37 °C in buffer containing 20 mM CAPS buffer, pH 1 1, + 10 mM DDT, + 0.1% 2- mercaptoethanol, for 2 hrs. The solution was centrifuged at 27,000 x g for 10 min. at 37 °C and the supernatant treated with 0.5% (w/v) TCPK treated trypsin (Sigma). This solution was incubated with mixing for an additional 1 hr. at room temperature, filtered, then loaded onto a Pharmacia Mono Q 1010 column equilibrated with 20 mM CAPS pH 10.5. After washing the loaded column with 2 column volumes of buffer, the truncated toxin was eluted using a linear gradient of 0 to 0.5 M NaCl in 20 mM CAPS in 15 column volumes at a flow rate of 1.0 ml/min. Purified trypsin truncated Cry proteins eluted at about 0.2-0.3 M NaCl. The purity of the proteins was checked by SDS PAGE and with visualization using Coomassie brilliant blue dye. In some cases, the combined fractions of the purified toxin were concentrated and loaded onto a Superose 6 column (1.6 cm dia., 60 cm long), and further purified by size exclusion chromatography. Fractions comprising a single peak of the monomeric molecular weight were combined, and concentrated, resulting in a preparation more than 95% homogeneous for a protein having a molecular weight of about 60,000 kDa.

Processing of Vip3Abl was achieved in a similar manner starting with the purified full length 85 kDa protein (DIG-307). The protein (12 mg) was dialyzed into 50 mM sodium phosphate buffer, pH 8.4, then processed by adding 1 mg of solid trypsin and incubating for 1 hrs. at room temperature. The solution was loaded onto a MonoQ anion exchange column (1 cm dia., 10 cm. long), and eluted with a linear gradient of NaCl from 0 to 500 mM in 20 mM sodium phosphate buffer, pH 8.4 over 7 column volumes. Elution of the protein was monitored by SDS-PAGE. The major processed band had a molecular weight of 65 kDa, as determined by SDS-PAGE using molecular weight standards for comparison. Example 2 - Iodination of CrylCa core toxin protein

Previous work indicated that CrylCa was very difficult to radiolabel using traditional methods, although in a select few cases it would radiolabel and function well in a receptor binding assay. We decided to radiolabel CrylCa using ¹²⁵I radiolabeled fluorescein-5-maleimide, which is a method that has worked to actively radiolabel Cry 1 Fa (Prov. 69919). Iodination of fluroescein-5- malemide and subsequent conjugation of this radiolabeled chemical with CrylCa results in cysteine specific radiolabeling of the protein. Such labeling procedure is thus highly specific in the residues that are labeled. The CrylCa core toxin segment (residues 29-619) contains two cysteine amino acid residues, at positions 210 and 438. Palmer et al. (1997) demonstrated that the phenyl rings of fluorescein-5- maleimide can be radio-iodinated and then reacted with proteins that contain sulfhydryl groups (e.g. as provided by free cysteine residues), resulting in alkylation of the free cysteines in the protein, and thus providing a radioactively labeled protein. The trypsin- truncated CrylCa core toxin contains two cysteine residues and thus provides a substrate for alkylation and radiolabeling of the protein at these two (specific) sites.

Fluorescein-5-maleimide (F5-M) was dissolved to 10 mM in DMSO (Dimethyl Sulfoxide), then diluted to 1 mM in phosphate buffered saline (PBS; 20 mM sodium phosphate, 0.15 M NaCl, pH7.5), as determined by the molar extinction coefficient of F 5- M (68,000 M^cm^"1). To a 100 μΐ_^ solution of PBS containing two Pierce Iodination Beads (Thermo Fisher Scientific), 1.0 mCi of Na¹²⁵I was added behind lead shielding. The solution was allowed to mix at room temperature for 5 min, then 10 μΐ. of the 1 mM F 5-M solution were added. After reacting for 10 min, the solution was removed from the iodination reaction by pipetting and 2 μg of highly purified trypsin-truncated CrylCa core toxin protein in PBS were added to the solution. The protein was incubated at 4° with the iodinated F 5-M solution for 48 hrs, when the reaction was terminated by adding β- mercaptoethanol to 14 mM final concentration. The reaction mixture was added to a Zebra™ spin column (Invitrogen) equilibrated in 20 mM CAPS, 150 mM KC1, pH9, and centrifuged at 1500 x g for 2 min to separate non-reacted iodinated dye from the protein. The ¹²⁵I radiolabeled fluorescein-CrylCa core toxin protein was counted in a gamma counter to determine its specific radioactivity, assuming 80% recovery of the input toxin protein.

The specific activity of the radiolabeled CrylCa core toxin protein was approximately 6.8 μθ/μζ protein. The radiolabeled protein was also characterized by SDS- PAGE and visualized by phosphor-imaging to validate that the radioactivity measured was covalently associated with the CrylCa core toxin protein. Coomassie stained SDS-PAGE gels were imaged by wrapping them in Mylar™ film (12 μιη thick), and exposing them under a Molecular Dynamics (Sunnyvale, CA) storage phosphor screen (35 cm x 43 cm) for 1 hour. The plates were developed using a Molecular Dynamics Storm 820 phosphor- imager and the image analyzed using ImageQuant™ software. Some radioactivity was detectable in the gel region well below the CrylCa core toxin protein band (i.e. fragments smaller than the CrylCa core toxin protein at about 10 kDa in size and lower). These radioactive contaminants likely represent small peptides probably associated in the truncated CrylCa protein due to the action of the trypsin used to cleave the protein to its core structure.

Example 3 - Competitive binding assays to BBMVs from S. frugiperda with core toxin proteins of CrylCa and Vip3Ab.

Homologous and heterologous competition binding assays were conducted using

150 μg/mL BBMV protein and 2 nM of the 1251-radiolabeled CrylCa core toxin protein.

Concentrations of the homologous competitive non-radiolabeled CrylCa core toxin protein added to the reaction mixture was 0.1, 1, 10, 100, and 1000 nM. The heterologous trypsin truncated Vip3Ab protein was tested at 10 and 1,000 nM and the proteins were added at the same time as the radioactive CrylCa core toxin protein to assure true binding competition.

Incubations were carried out for 1 hr at 28° and the amount of 1251-labeled CrylCa core toxin protein unbound to the BBMV's (that is, not bound to an insect receptor protein) is separated from bound protein by centrifugation of the BBMV mixture at 16,000 x g for 8 min, and removing the supernatant from the resulting pellet. The pellet is washed three times with ice cold binding buffer (PBS; 11.9 mM Na₂HP0₄, 137 mM NaCl, 2.7 mM KC1, pH7.4 plus 0.1% bovine serum albumin; Sigma- Aldrich, St. Louis, MO) to completely remove any remaining unbound ¹²⁵I labeled CrylCa. The bottom the centrifuge tube was cut out and the protein pellet contained within this section placed in a 13 x 100 mm glass culture tube and counted in a gamma counter for 10 minutes to obtain the amount of bound radioactivity contained the pellet fraction. The amount of radioactivity in the bound protein fraction provides an indication of the amount of Cry protein bound to the insect receptor

(total binding). Non-specific binding was represented by the counts obtained in the pellet in the presence of 1,000 nM of non-radiolabeled CrylCa core toxin protein. The amount of radiolabeled CrylCa specifically bound to the BBMV (specific binding) was measured by subtracting the level of total binding from non specific binding. One hundred percent total binding was considered to be the amount of binding in the absence of any competitor Cry 1 Fa core toxin protein. The data is expressed as percent of specific bound ¹²⁵I CrylCa versus concentration of competitive unlabeled ligand.

Example 4 - Summary of Results

The results (Figure 1) show that the homologous unlabeled CrylCa protein effectively displaced the radiolabeled CrylCa core toxin protein from specifically binding to the BBMV proteins in a dose dependent manner. Vip3Ab did not displace bound 1251- labeled CrylCa core toxin protein from its receptor protein(s) at either of the concentrations shown (10 or 1,000 nM). The highest concentration of Vip3Ab tested (1,000 nM) represents 500-fold greater concentration than the radiolabeled CrylCa used in the assay, demonstrating that Vip3 Ab does not effectively compete with the binding of radiolabeled CrylCa in S. frugiperda BBMV.

Figure 1 is a dose response curve for the displacement of ¹²⁵I radiolabeled fluorescein-5-maleimide trypsin-truncated CrylCa in BBMV's from S. frugiperda (FAW) larvae. The figure shows the ability of non-labled CrylCa (·) to displace the labeled CrylCa in a dose dependent manner in the range from 0.1 to 1,000 nM. The chart plots the percent of specifically bound labeled CrylCa (total bound minus non-specific bound) versus the concentration of the non-radiolabeled ligands added. The inability of non radiolabeled Vip3Abl (A) at 10 and 1,000 nM to displace the specifically bound radiolabeled CrylCa is shown.

Reference List

Heckel,D.G., Gahan,L.J., Baxter,S.W., Zhao,J.Z., Shelton,A.M., Gould,F., and

Tabashnik,B.E. (2007). The diversity of Bt resistance genes in species of Lepidoptera. J Invertebr Pathol 95, 192-197.

Luo,K., Banks,D., and Adang,M.J. (1999). Toxicity, binding, and permeability analyses of four bacillus thuringiensis cryl delta-endotoxins using brush border membrane vesicles of spodoptera exigua and spodoptera frugiperda. Appl. Environ. Microbiol. 65, 457-464.

Palmer, M., Buchkremer, M, Valeva, A, and Bhakdi, S. Cysteine-specific radioiodination of proteins with fluorescein maleimide. Analytical Biochemistry 253, 175-179. 1997.

Ref Type: Journal (Full)

Sambrook,J. and Russell,D.W. (2001). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory).

Schlenz, M. L., Babcock, J. M., and Storer, N. P. Response of CrylF-resistant and

Susceptible European Corn Borer and Fall Armyworm Colonies to Cry 1A.105 and

Cryl2Ab2. DAI 0830, 2008. Indianapolis, Dow AgroSciences. Derbi Report.

Sheets, J. J. and Storer, N. P. Analysis of CrylAc Binding to Proteins in Brush Border Membrane Vesicles of Corn Earworm Larvae (Heleothis zea). Interactions with Cry IF Proteins and Its Implication for Resistance in the Field. DAI-0417, 1-26. 2001. Indianapolis, Dow AgroSciences.

Tabashnik,B.E., Liu,Y.B., Finson,N., Masson,L., and Heckel,D.G. (1997). One gene in diamondback moth confers resistance to four Bacillus thuringiensis toxins. Proc. Natl. Acad. Sci. U. S. A 94, 1640-1644.

Tabashnik,B.E., Malvar,T., Liu,Y.B., Finson,N., Borthakur,D., Shin,B.S., Park,S.FL, Masson,L., de Maagd,R.A., and Bosch,D. (1996). Cross-resistance of the diamondback moth indicates altered interactions with domain II of Bacillus thuringiensis toxins. Appl. Environ. Microbiol. 62, 2839-2844.

Tabashnik,B.E., Roush,R.T., Earle,E.D., and Shelton,A.M. (2000). Resistance to Bt toxins. Science 287, 42.

Wolfersberger,M.G. (1993). Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the gypsy moth (Lymantria dispar). Arch. Insect Biochem. Physiol 24, 139-147.

Xu,X., Yu,L., and Wu,Y. (2005). Disruption of a cadherin gene associated with resistance to CrylAc {delta} -endotoxin of Bacillus thuringiensis in Helicoverpa armigera. Appl Environ Microbiol 71, 948-954. Appendix A

List of delta-endotoxins - from Crickmore et al. website (cited in application)

Accession Number is to NCBI entry

Name Acc No. Authors Year Source Strain Comment

CrvlAal AAA22353 Schnepf et al 1985 Bt kurstaki HD 1

Crv l Aa2 AAA22552 Shibano et al 1985 Bt sotto

CrylAa.3 BAA00257 Shimizu et al 1988 Bt aizawai IPL7

Crv l Aa4 CAA31886 Masson et al 1989 Bt entomocidus

CrviAa.5 BAA04468 Udayasuriyan et al 1994 Bt Fu-2-7

Bt kurstaki NRD

CrvlA 6 AAA86265 Masson et al 1994

12

CrylAa? AAD46139 Osman et al 1999 Bt C 12

DNA sequence

CrylAa8 126149 Liu 1996

only

Bt dendrolimus

CrvlAa9 BAA77213 Nagamatsu et al 1999

T84A1

Bt kurstaki HD-1-

CrylAalO AAD55382 Hou and Chen 1999

02

CrylAal 1 CAA70856 Tounsi et al 1999 Bt kurstaki

Crv l Aal 2 AAP80146 Yao et al 2001 Bt Ly30

CrylAal 3 AAM44305 Zhong et al 2002 Bt sotto

Crv l Aal 4 AAP40639 Ren et al 2002 unpublished

CrylAal 5 AAY66993 Sauka et al 2005 Bt INT A Mol-12

Cry l Abl AAA22330 Wabiko et al 1986 Bt berliner 1715

CrylAb2 AAA22613 Thorne et al 1986 Bt kurstaki

Cry l Ab3 AAA22561 Geiser et al 1986 Bt kurstaki HD 1

CrylAb4 BAA00071 Kondo et al 1987 Bt kurstaki HD1

Cry l Ab5 CAA28405 Hofte et al 1986 Bt berliner 1715

Bt kurstaki NRD-

Crv l Ab6 AAA22420 Hefford et al 1987

12

CrylAb7 CAA31620 Haider & Ellar 1988 Bt aizawai IC1

CrylAb8 AAA22551 Oeda et al 1987 Bt aizawai IPL7

CrylAb9 CAA38701 Chak & Jen 1993 Bt aizawai HD133

CrylAblO A29125 Fischhoff et al 1987 Bt kurstaki HD1

DNA sequence

Cryl Abl l 112419 Ely & Tippett 1995 Bt A20

only

Cry lAbl 2 AAC64003 Silva-Werneck et al 1998 Bt kurstaki S93

CrylAbl3 AAN76494 Tan et al 2002 Bt c005

Meza-Basso &

CrylAbl4 AAG 16877 Native Chilean Bt

Theoduloz CrylAb 15 AAO 13302 Li et al 2001 Bt B-Hm- 16

CrylAbK) AAK55546 Yu et al 2002 Bt AC- 1 1

CrylAb 17 ^• AAT46415 Huang et al 2004 Bt WB9

CrylAbl S AA088259 Stobdan et al 2004 Bt

CrylAb 19 AAW3 1761 Zhong et al 2005 Bt X-2

CrylAb20 ABB72460 Liu et al 2006 BtC008

CrvlAb2 l ABS 18384 Swiecicka et al 2007 Bt IS5056

CrvlAb22 ABW87320 Wu and Feng 2008 BtS2491Ab

CrylAb- uncertain

AAK14336 Nagarathinam et al 2001 Bt kunthala RX24

like sequence

CrylAb- uncertain

AAK14337 Nagarathinam et al 2001 Bt kunthala RX28

!ike sequence

CrylAb- uncertain

AAK14338 Nagarathinam et al 2001 Bt kunthala RX27

like sequence

Crv l Ab- insufficient

ABG88858 Lin et al 2006 Bt Iy4a3

like sequence

CrylAc l AAA2233 1 Adang et al 1985 Bt kurstaki HD73

CrylAc2 AAA22338 Von Tersch et al 1991 Bt kenyae

CrylAc3 CAA38098 Dardenne et al 1990 Bt BTS89A

Bt kurstaki

Cry lAc4 AAA73077 Feitelson 1991

PS85A1

Bt kurstaki

CrylAcS AAA22339 Feitelson 1992

PS81 GG

Bt kurstaki NRD-

CrylAc6 AAA86266 Masson et al 1994

12

CrylAc7 AAB46989 H err era et al 1994 Bt kurstaki HD73

Crv l Ac8 AAC44841 Omolo et al 1997 Bt kurstaki HD73

CrylAc9 AAB49768 Gleave et al 1992 Bt DSIR732

Bt kurstaki YBT-

Cry 1 Ac 10 CAA05505 Sun 1997

1520

Makhdoom &

CrylAc l 1 CAA 10270 1998

iazuddin

DNA sequence

Crv lAc l2 112418 Ely & Tippett 1995 Bt A20

only

CrylAc l 3 AAD38701 Qiao et al 1999 Bt kurstaki HD 1

CrylAc l 4 AAO06607 Yao et al 2002 Bt Ly30

CrylAc l 5 AAN07788 Tzeng et al 2001 Bt from Taiwan

CrylAc l 6 AAU87037 Zhao et al 2005 Bt H3

CrylAc l 7 AAX 18704 Hire et al 2005 Bt kenyae HD549

CrylAc l 8 AAY88347 Kaur & Allam 2005 Bt SK-729

CrylAc 9 ABD37053 Gao et al 2005 Bt C-33

CrylAc20 ABB89046 Tan et al 2005

CrylAc21 AAY66992 Sauka et al 2005 ΓΝΤΑ Mol-12

CrylAc22 ABZ01836 Zhang & Fang 2008 Bt W015- l

CrylAc23 CAO30431 Kashyap et al 2008 Bt CrylAc24 ABL01535 Arango et al 2008 Bt 146- 158-01 CrylAc25 FJ513324 No NCBI link

Guan Peng et al 2008 Bt Tm37-6

July 09 No NCBI link

CrylAc26 FJ617446 Guan Peng et al 2009 Bt Tm41-4

July 09

No NCBI link Cry 1 Ac27 FJ617447 Guan Peng et al 2009 Bt Tm44- 1 B

July 09

Crv lAc28 ACM90319 Li et al 2009 Bt Q-12

Cry l Adl AAA22340 Feitelson 1993 Bt aizawai PS81I

CrylAd2 CAA01880 Anonymous 1995 Bt PS81RRl

Crv l Ael AAA22410 Lee & Aronson 1991 Bt alesti

CrylAfl AAB82749 Kang et al 1997 Bt NT0423

Cry l Agl AAD46137 Mustafa 1999

CrylAh l AAQ 14326 Tan et al 2000

Crv l Ah2 ABB76664 Qi et al 2005 Bt alesti

Cryl Ai l AA039719 Wang et al 2002

Cry 1 A- uncertain

AA 14339 Nagarathinam et al 2001 Bt kunthala nags3

like sequence

Bt thuringiensis

CrylBal CAA29898 Brizzard & Whiteley 1988

HD2

Cry l Ba2 Bt entomocidus

CAA65003 Soetaert 1996

HD1 10

CrylBa3 AAK63251 Zhang et al 2001

CrylBa4 AA 51084 Bt entomocidus

Nathan et al 2001

HD9

CrylBa5 ABO20894 Song et al 2007 Bt sfw-12

Crv l Ba6 ABL60921 Martins et al 2006 Bt S601

CrylBbl AAA22344 Donovan et al 1994 Bt EG5847

Cry l Bel CAA86568 Bishop et al 1994 Bt morrisoni

Crv l Bdl AAD 10292 Bt wuhanensis

uo et al 2000

HD525

CrylBd2 AAM93496 Isakova et al 2002 Bt 834

Cryl Bel AAC32850 Payne et al 1998 Bt PS158C2

CrylBe2 AAQ52387 Baum et al 2003

CrylBe3 FJ716102 No NCBI link

Xiaodong Sun et al 2009

July 09

CrylBfl CAC50778 Arnaut et al 2001

Cry lBO AAQ52380 Baum et al 2003

CrylBgl AAO39720 Wang et al 2002

CrylCal Bt entomocidus

CAA30396 Honee et al 1988

60.5

Cryl Ca2 CAA31951 Sanchis et al 1989 Bt aizawai 7.29

CrylCa3 AAA22343 Feitelson 1993 Bt aizawai PS81I

Bt entomocidus

CrylCa4 CAA01886 Van Mellaert et al 1990

HD1 10 Cryl Ca.5 CAA65457 Strizhov 1996 Bt aizawai 7.29 CrylCa6 AAF37224 Yu et al 2000 Bt AF-2

CrylCa.7 AAG50438 Aixing et al 2000 Bt J8

CrylCaS AAM00264 Chen et al 2001 Bt c002

CrylCa9 AAL79362 Kao et al 2003 Bt GlO-OlA

CrylCalO AAN 16462 Lin et al 2003 Bt E05-20a

Cry 1 Cal l AAX53094 Cai et al 2005 Bt C-33

Cryl Cbl M97880 Kalman et al DNA sequence

1993 Bt galleriae HD29

only

Cry l Cb2 AAG35409 Song et al 2000 Bt cOOl

CrylCb3 ACD50894 Huang et al 2008 Bt 087

Crv l Cb- Thammasittirong et

AAX63901 insufficient like al 2005 Bt TA476-l

sequence

Cry 1 Dal CAA38099 Hofte et al 1990 Bt aizawai HD68

CrylDa2 176415 Payne & Sick 1997 DNA sequence only

Crv l Db l CAA80234 Lambert 1993 Bt BTS00349A

CrylDb2 AAK48937 Li et al 2001 Bt B-Pr-88

Cry 1 Del ABK35074 Lertwiriyawong et al 2006 Bt JC291

CrylEal CAA37933 Visser et al 1990 Bt kenyae 4F 1

Crv l Ea2 CAA39609 Bosse et al 1990 Bt kenyae

CrylEa3 AAA22345 Feitelson 1991 Bt kenyae PS8 IF

CrylEa4 AAD04732 Barboza-Corona et Bt kenyae LBIT- al 1998

147

CrylEaS A15535 Botterman et al 1994 DNA sequence only

Crv lEa6 AAL50330 Sun et al 1999 Bt YBT-032

Crv l Ea7 AAW72936 Huehne et al 2005 Bt JC190

Cry lEaS ABX1 1258 Huang et al 2007 Bt HZM2

CrvlEbl AAA22346 Feitelson Bt aizawai

1993

PS81A2

Cr lFal AAA22348 Chambers et al Bt aizawai

1991

EG6346

Crv i Fa2 AAA22347 Feitelson 1993 Bt aizawai PS81I

Crv l Fbl CAA80235 Lambert 1993 Bt BTS00349A

Crv l Fb2 BAA25298 Masuda & Asano Bt morrisoni

1998

INA67

CrylFb3 AAF21767 Song et al 1998 Bt morrisoni

CrylFb4 AAC 10641 Payne et al 1997

CrylFbS AA013295 Li et al 2001 Bt B-Pr-88

CrylFb6 ACD50892 Huang et al 2008 Bt 012

CrylFb7 ACD50893 Huang et al 2008 Bt 087

Cry 1 Gal CAA80233 Lambert 1993 Bt BTS0349A

CrylGa2 CAA70506 Shevelev et al 1997 Bt wuhanensis

CrylGbl AAD 10291 Kuo & Chak 1999 Bt wuhanensis HD525

CrylGb2 AA013756 Li et al 2000 Bt B-Pr-88

CrylGc AAQ52381 Baum et al 2003

Cry 1 Hal CAA80236 Lambert 1993 Bt BTS02069AA

Bt morrisoni

Cr lHbl AAA79694 oo et al 1995

BF190

AAF01213 Srifahetal 1999 BUC291 insufficient sequence

Cryllal CAA44633 Tailor et al 1992 Bt kurstaki

Crylla2 AAA22354 Gleaveetal 1993 Bt kurstaki

Crylla3 AAC36999 Shin et al 1995 Bt kurstaki HD1

Cry! AAB00958 Kostichka et al 1996 Bt AB88

Crylla5 CAA70124 Selvapandiyan 1996 Bt61

Crylia6 AAC26910 Zhongetal 1998 Bt kurstaki S101

CryJlaJ AAM73516 Porcar et al 2000 Bt

Crylla8 AA 66742 Songetal 2001

Crylla9 AAQ08616 Yao et al 2002 Bt Ly30

CryllalO AAP86782 Espindola et al 2003 Bt thuringiensis

Cryllal 1 CAC85964 Tounsi et al 2003 Bt kurstaki BNS3

Cryllal2 AAV53390 Grossi de Sa et al 2005 Bt

Cryllal 3 ABF83202 Martins et al 2006 Bt

Cryllal4 ACG63871 Liu & Guo 2008 Btll

No NCBI link

Cry Hal 5 FJ617445 Guan Peng et al 2009 Bt E-1B

July 2009 No NCBI link

Cryllal6 FJ617448 Guan Peng et al 2009 Bt E-1A

July 2009

Bt entomocidus

Cryllbl AAA82114 Shinetal 1995

BP465

Cryllb2 ABW88019 Guan et al 2007 Bt PP61

Cr llb3 ACD75515 Liu & Guo 2008 Bt GS8

Cryllcl AAC62933 Osmanetal 1998 BtC18

CrylTc2 AAE71691 Osmanetal 2001

Cjylldl AAD44366 Choi 2000

Cryllel AAG43526 Song et al 2000 Bt BTC007

Cryllfl AAQ52382 Baum et al 2003

insufficient

CjylHike AAC31094 Payne et al 1998

sequence insufficient

Cry 1 Hike ABG88859 Lin & Fang 2006 Bt Iy4a3

sequence

CrylJal AAA22341 Donovan 1994 Bt EG5847

Von Tersch &

CrylJ l AAA98959 Bt EG5092

Gonzalez

CrylJcl AAC31092 Payne et al 1998

CrylJc2 AAQ52372 Baum et al 2003

CrylJdl CAC50779 Amautetal 2001 Bt Cr lKal AAB00376 Koo et al Bt morrisoni

1995

BF190

Cry 1 Lai AAS60191 Je et al 2004 Bt kurstaki Kl

Crvl -like AAC31091 Payne et al 1998 insufficient sequence

Cry2Aal AAA22335 Donovan et al 1989 Bt kurstaki

Cry2Aa2 AAA83516 Widner & Whiteley 1989 Bt kurstaki HD1

Cry2Aa3 D86064 Sasaki et al 1997 Bt sotto DNA sequence only

Cry2Aa4 AAC04867 Misra et al 1998 Bt kenyae HD549

Crv2Aa5 CAA 10671 Yu & Pang 1999 Bt SL39

Crv2Aa6 CAA 10672 Yu & Pang 1999 Bt YZ71

Crv2Aa7 CAA 10670 Yu & Pang 1999 Bt CY29

Crv2Aa8 AA013734 Wei et al 2000 Bt Dongbei 66

Crv2Aa9 AAO13750 Zhang et al 2000

Crv2Aa1 ( ) AAQ04263 Yao et al 2001

Crv2Aal l AA052384 Baum et al 2003

Crv2Aal 2 AB183671 Tan et al 2006 Bt Rpp39

Crv2Aah^" 1 ABL01536 Arango et al 2008 Bt 146-158-01

Crv2Aal 4 ACF04939 Hire et al 2008 Bt HD-550

Crv2Abl AAA22342 Widner & Whiteley 1989 Bt kurstaki HD1

Crv2Ab2 CAA39075 Dankocsik et al 1990 Bt kurstaki HD1

Crv2Ab3 AAG36762 Chen et al 1999 Bt BTC002

Crv2Ab4 AA013296 Li et al 2001 Bt B-Pr-88

Crv2Ab5 AAQ04609 Yao et al 2001 Bt ly30

Crv2Ab6 AAP59457 Wang et al 2003 Bt WZ-7

Cry2Ab7 AAZ66347 Udayasuriyan et al 2005 Bt 14-1

Crv2Ab8 ABC95996 Huang et al 2006 Bt WB2

Crv2Ab9 ABC74968 Zhang et al 2005 Bt LLB6

Cry2AbiO EF 157306 Lin et al 2006 Bt LyD

Crv2Ab1 1 CAM84575 Saleem et al 2007 Bt CMBL-BT1

Crv2Abl2 ABM21764 Lin et al 2007 Bt LyD

Crv2Abl 3 ACG761 70 Zhu et al 2008 Bt ywc5-4

Crv2Abl4 A G761 71 Zhu et al 2008 Bt Bts

Crv2Acl CAA40536 Aronson 1991 Bt shanghai SI

Crv2Ac2 AAG35410 Song et al 2000

Crv2Ac3 AAQ52385 Baum et al 2003

Crv2Ac4 ABC95997 Huang et al 2006 Bt WB9

Cry2Ac5 ABC74969 Zhang et al 2005

Cry2Ac6 ABC74793 Xia et al 2006 Bt wuhanensis

Cry2Ac7 CAL 18690 Saleem et al 2008 Bt SBSBT-1

Crv2Ac8 CAM09325 Saleem et al 2007 Bt CMBL-BT1

Cry2Ac9 CAM09326 Saleem et al 2007 Bt CMBL-BT2

Crv2Acl O ABN15104 Bai et al 2007 Bt QCL-1 Crv2Acl l CAM83895 Saleem et al 2007 Bt HD29

Cry2Ac i2 CAM83896 Saleem et al 2007 Bt CMBL-BT3

Cry2Adl AAF09583 Choi et al 1999 Bt BR30

Cry2Ad2 ABC86927 Huang et al 2006 Bt WB10

Cry2Ad3 CAK29504 Saleem et al 2006 Bt 5_2AcT(l)

Cry2Ad4 CAM32331 Saleem et al 2007 Bt CMBL-BT2

Crv2Ad5 CA078739 Saleem et al 2007 Bt HD29

Crv2Ael AAQ52362 Baum et al 2003

Crv2Afl ABO30519 Beard et al 2007 Bt C81

Cxy2Ag ACH91610 Zhu et al 2008 Bt JF19-2

Cry2Ah No NCBI link

EU939453 Zhang et al 2008 Bt

July 09

Crv2Ah2 ACL80665 Zhang et al 2009 Bt BRC-ZQL3

Cry2Ai No NCBI link

FJ788388 Udayasuriyan et al 2009 Bt

July 09

Crv3Aal AAA22336 Herrnstadt et al 1987 Bt san diego

Crv3Aa2 AAA22541 Sekar et al 1987 Bt tenebrionis

Crv3Aa3 CAA68482 Hofte et al 1987

Cry3Aa4 AAA22542 McPherson et al 1988 Bt tenebrionis

Bt morrisoni

Crv3Aa5 AAA50255 Donovan et al 1988

EG2158

Crv3Aa6 AAC43266 Adams et al 1994 Bt tenebrionis

Crv3Aa7 CAB4141 1 Zhang et al 1999 Bt 22

Cry3Aa8 AAS79487 Gao and Cai 2004 Bt YM-03

Cry3Aa9 AAW05659 Bulla and Candas 2004 Bt UTD-001

Crv3Aal0 AAU2941 1 Chen et al 2004 Bt 886

Bt tenebrionis

Cry3Aal l AAW82872 Kurt et al 2005

Mm2

Crv3Aal2 ABY49136 Sezen et al 2008 Bt tenebrionis

Crv3Bal CAA34983 Sick et al 1990 Bt tolworthi 43 F

Crv3Ba2 CAA00645 Peferoen et al 1990 Bt PGSI208

Crv3Bbl AAA22334 Donovan et al 1992 Bt EG4961

Crv3Bb2 AAA74198 Donovan et al 1995 Bt EG 144

DNA sequence

Crv3Bb3 115475 Peferoen et al 1995

only

Bt kurstaki

Crv3Cal CAA42469 Lambert et al 1992

BtI109P

Cry4Aal CAA68485 Ward & Ellar 1987 Bt israelensis

Bt israelensis

Crv4Aa2 BAA00179 Sen et al 1988

HD522

Cry4Aa3 CAD30148 Berry et al 2002 Bt israelensis

Cry4A- AAY96321 insufficient

Mahalakshmi et al 2005 Bt LDC-9 like sequence

Chungjatpornchai et Bt israelensis

Crv4Bal CAA30312 1988

al 4Q2-72 Cry4Ba.2 CAA301 14 Tungpradubkul et al 1988 Bt israelensis

Crv4Ba3 AAA22337 Yamamoto et al 1988 Bt israelensis

Cry4Ba4 BAA00178 Sen et al Bt israelensis

1988

HD522

Crv4Ba5 CAD30095 Berry et al 2002 Bt israelensis

Crv4Ba-

ABC47686 Mahalakshmi et al insufficient like 2005 Bt LDC-9

sequence

Cry4Cal EU646202 Shu et al No NCBI link

2008

July 09

Cry4Cbl FJ403208 Jun & Furong No NCBI link

2008 Bt HS 18-1

July 09

Cry4Cb2 FJ597622 Jun & Furong No NCBI link

2008 Bt Ywc2-8

July 09

Cry4Ccl FJ403207 Jun & Furong No NCBI link

2008 Bt MC28

July 09

CrySAal AAA67694 Narva et al Bt darmstadiensis

1994

PS 17

Crv5Abl AAA67693 Narva et al Bt darmstadiensis

1991

PS 17

Cry5Acl 134543 Payne et al DNA sequence

1997

only

Crv5Adl ABQ82087 Lenane et al 2007 Bt L366

CrvSBal AAA68598 Foncerrada & Narva 1997 Bt PS86Q3

Crv5Ba2 ABW88932 Guo et al 2008 YBT 1518

Crv6Aal AAA22357 Narva et al 1993 Bt PS52Al

Crv6Aa2 AAM46849 Bai et al 2001 YBT 1518

Crv6Aa3 ABH03377 Jia et al 2006 Bt 96418

Crv6Bal AAA22358 Narva et al 1991 Bt PS69D1

Crv7Aal AAA22351 Lambert et al Bt galleriae

1992

PGSI245

Cry7Abl AAA21 120 Narva & Fu 1994 Bt dakota HD51 1

Crv7Ab2 AAA21 121 Bt kumamotoensis

Narva & Fu 1994

867

Crv7Ab3 ABX24522 Song et al 2008 Bt WZ-9

Cry7Ab4 EU380678 Shu et al No NCBI link

2008 Bt

July 09

Ci 7Ab5 ABX79555 Bt monterrey GM-

Aguirre-Arzola et al 2008

33

Crv7Ab6 ACI44005 Deng et al 2008 Bt HQ 122

Cry7Ab7 FJ940776 Wang et al No NCBI link

2009

Sept 09

Cry7Ab8 GU145299 Feng Jing No NCBI link

2009

Nov 09

Crv7Bal ABB70817 Zhang et al 2006 Bt huazhongensis

Crv7Cal ABR67863 Gao et al 2007 Bt BTH-13

Crv7Dal ACQ99547 Yi et al 2009 Bt LH-2 CrySAal AAA21 117 Narva & Fu 1992 Bt kumamotoensis

No NCBI link

Cry8Abl EU044830 Cheng et al 2007 Bt B-JJX

July 09

Crv8Bal AAA21 1 18 Narva & Fu 1993 Bt kumamotoensis

CrvSBbl CAD57542 Abad et al 2002

Crv8Bcl CAD57543 Abad et al 2002

CrvSCal Bt japonensis

AAA21 119 Sato et al. 1995

Buibui

Crv8Ca2 AAR98783 Shu et al 2004 Bt HBF- 1

No NCBI link

Cry8Ca3 EU625349 Du et al 2008 Bt FTL-23

July 09

CrvSDal BAC07226 Asano et al 2002 Bt galleriae

Crv8Da2 BD133574 DNA sequence

Asano et al 2002 Bt

only

DNA sequence

Crv8Da3 BD133575 Asano et al 2002 Bt

only

CrvSDbi BAF93483 Yamaguchi et al 2007 Bt BBT2-5

Crv8Eal AAQ73470 Fuping et al 2003 Bt 185

Cry8Ea2 No NCBI link

EU047597 Liu et al 2007 Bt B-DLL

July 09

Cry8Fal AAT48690 Shu et al 2004 Bt 185 also AAW81032

Crv8Gal AAT46073 Shu et al 2004 Bt HBF- 18

Cry8Ga2 ABC42043 Yan et al 2008 Bt 145

No NCBI link

Cry8Ga3 FJ198072 Xiaodong et al 2008 Bt FCD1 14

July 09

Cry8Hal No NCBI link

EF465532 Fuping et al 2006 Bt 185

July 09

Cry8Ial No NCBI link

EU381044 Yan et al 2008 Bt su4

July 09

Cry8Jal No NCBI link

EU625348 Du et al 2008 Bt FPT-2

July 09

No NCBI link

Cry8Kal FJ422558 Quezado et al 2008

July 09

Crv8Ka2 ACN87262 Noguera & Ibarra 2009 Bt kenyae

DNA sequence

Crv8-like FJ770571 Noguera & Ibarra 2009 Bt canadensis

only

Crv8-like ABS53003 Mangena et al 2007 Bt

Crv9Aal CAA41122 Shevelev et al 1991 Bt galleriae

Crv9Aa2 CAA41425 Gleave et al 1992 Bt DSIR517

No NCBI link

Cry9Aa3 GQ249293 Su et al 2009 Bt SC5(D2)

July 09

Cry9Aa4 No NCBI link

GQ249294 Su et al 2009 Bt T03C001

July 09

Crv9Aa incomplete

AAQ52376 Baum et al 2003

like sequence

Crv9Bal CAA52927 Shevelev et al 1993 Bt galleriae Crv9Bbl AAV28716 Silva-Wemeck et al 2004 Bt japonensis

Crv9Cal CAA85764 Lambert et al 1996 Bt tolworthi

Cry9Ca2 AAQ52375 Baum et al 2003

Bt japonensis

Crv9Dal BAA 19948 Asano 1997

N141

Crv9Da2 AAB97923 Wasano & Ohba 1998 Bt japonensis

No NCBI link

Cry9Da3 GQ249295 Su et al 2009 Bt T03B001

July 09

No NCBI link

Cry9Da4 GQ249297 Su et al 2009 Bt T03B001

July 09

Bt kurstaki

Crv9Dbl AAX78439 Flannagan & Abad 2005

DP 1019

Bt aizawai SSK-

Crv9Eal BAA34908 Midoh & Oyama 1998

10

Crv9Ea2 AAO12908 Li et al 2001 Bt B-Hm-16

Crv9Ea3 ABM21765 Lin et al 2006 Bt lyA

Crv9Ea4 ACE88267 Zhu et al 2008 Bt ywc5-4

Crv9Ea5 ACF04743 Zhu et al 2008 Bts

Crv9Ea6 ACG63872 Liu & Guo 2008 Bt 1 1

No NCBI link

Cry9Ea7 FJ380927 Sun et al 2008

July 09

No NCBI link

Cry9Ea8 GQ249292 Su et al 2009 GQ249292

July 09

Crv9Eb l CAC50780 Arnaut et al 2001

No NCBI link

Ciy9Eb2 GQ249298 Su et al 2009 Bt T03B001

July 09

Crv9Ecl AAC63366 Wasano et al 2003 Bt galleriae

Bt kurstaki

Crv9Ed l AAX78440 Flannagan & Abad 2005

DP 1019

No NCBI link

Cry9Eel GQ249296 Su et al 2009 Bt T03B001

Aug 09 insufficient

Cry9-like AAC63366 Wasano et al 1998 Bt galleriae

sequence

CrylOAal AAA22614 Thorne et al 1986 Bt israelensis

Bt israelensis DNA sequence

CrylOAa2 E00614 Aran & Toomasu 1996

ONR-60A only

CrylOAa.3 CAD30098 Berry et al 2002 Bt israelensis

Cry 1 OA- incomplete

DQ 167578 Mahalakshmi et al 2006 Bt LDC-9

like sequence

Crv l l Aal AAA22352 Donovan et al 1988 Bt israelensis

Cryl lAa2 AAA2261 1 Adams et al 1989 Bt israelensis

Cryl lAa3 CAD30081 Berry et al 2002 Bt israelensis

Cryl lAa- incomplete

DQ 166531 Mahalakshmi et al 2007 Bt LDC-9

like sequence

Cryl lBal CAA60504 Delecluse et al 1995 Bt jegathesan 367 Cry 1 IBM AAC97162 Orduz et al 1998 Bt medellin

Cryl2Aal AAA22355 Narva et al 1991 Bt PS33F2

CryOAal AAA22356 Narva et al 1992 Bt PS63B

Cryl4Aal AAA21516 Narva et al 1994 Bt sotto PS80JJ1

CrylSAal AAA22333 Brown & Whiteley 1992 Bt thompsoni

Cryl6Aal CAA63860 Barloy et al 1996 Cb malaysia CHU

Cryl7Aal CAA67841 Barloy et al 1998 Cb malaysia CHU

Cryl 8Aal CAA67506 Zhang et al Paenibacillus

1997

popilliae

Cryl 8Bal AAF89667 Patel et al Paenibacillus

1999

popilliae

CryiSCal AAF89668 Patel et al Paenibacillus

1999

popilliae

Cryl9Aa1 CAA68875 Rosso & Delecluse 1996 Bt jegathesan 367

Cry l 9Ba1 BAA32397 Hwang et al 1998 Bt higo

Crv20Aa1 AAB93476 Lee & Gill 1997 Bt fukuokaensis

Cry20Ba1 ACS93601 Noguera & Ibarra 2009 Bt higo LBIT-976

Cry20-jike GO 144 Yi et al 2009 Bt Y-5 DNA sequence only

COi21 Aal 132932 Payne et al 1996 DNA sequence only

Crv21Aa2 166477 Feitelson 1997 DNA sequence only

Cry21Ba l BAC06484 Sato & Asano 2002 Bt roskildiensis

Cjy22Aal 134547 Payne et al 1997 DNA sequence only

Cry22Aa2 CAD43579 Isaac et al 2002 Bt

Crv22Aa3 ACD93211 Du et al 2008 Bt FZ-4

Crv22Ah l AAK50456 Baum et al 2000 Bt EG4140

Crv22Ab2 CAD43577 Isaac et al 2002 Bt

Cry22Bal CAD43578 Isaac et al 2002 Bt

3Aal AAF76375 Donovan et al 2000 Bt Binary with

Cry37Aal

Crv24Aal AAC61891 Kawalek and Gill 1998 Bt jegathesan

Cry24Bal BAD32657 Ohgushi et al 2004 Bt sotto

Crv24Cal CAJ43600 Beron & Salerno 2005 Bt FCC-41

Cry25Aal AAC61892 Kawalek and Gill 1998 Bt jegathesan

Cry26AaI Wojciechowska et

AAD25075 Bt finitimus B- al 1999

1 166

Crv27Aal BAA82796 Saitoh 1999 Bt higo

Cry28Aal AAD24189 Wojciechowska et al 1999 Bt finitimus B- 1 161

Cry28Aa2 AAG00235 Moore and Debro 2000 Bt finitimus

Cry29Aal CAC80985 Delecluse et al 2000 Bt medellin

Crv30Aal CAC80986 Delecluse et al 2000 Bt medellin Crv30Bal BAD00052 Ito et al 2003 Bt entomocidus

CrySOCal BAD67157 Ohgushi et al 2004 Bt sotto

Cry30Ca2 ACU24781 Sun and Park 2009 Bt jegathesan 367

Cry30Dal EF095955 No NCBI link

2006 Bt Y41

July09

Bt aizawai BUT

Crv30Dbi BAE80088 Kishida et al 2006

14

Cry30Eai ACC95445 Fang et al 2007 Bt S2160-1

Cry30Ea2 FJ499389 Jun et al No NCBI link

2008 Bt Ywc2-8

July09

Crv30Fal ACI22625 Tan et al 2008 Bt MC28

Crv30Gal ACG60020 Zhu et al 2008 Bt HS 18-1

Crv31 Aal BAB 11757 Saitoh & Mizuki 2000 Bt 84-HS-l-l l

Crv31 Aa2 AAL87458 Jung and Cote 2000 Bt M15

Crv 1 Aa3 BAE79808 Uemori et al 2006 Bt B0195

Crv31Aa4 BAF32571 Yasutake et al 2006 Bt 79-25

Crv31 Aa5 BAF32572 Yasutake et al 2006 Bt 92-10

Crv3 1Abl BAE79809 Uemori et al 2006 Bt B0195

Crv3 1 Ab2 BAF32570 Yasutake et al 2006 Bt 31 -5

Crv31 Ac1 BAF34368 Yasutake et al 2006 Bt 87-29

Balasubramanian et

Crv32Aal AAG3671 1 2001

al Bt yunnanensis

Cry32Bal BAB78601 Takebe et al 2001 Bt

Crv32Cal BAB78602 Takebe et al 2001 Bt

Crv32Dal BAB78603 Takebe et al 2001 Bt

Cry33Aal AAL26871 Kim et al 2001 Bt dakota

Crv34Aal AAG50341 Binary with

Ellis et al 2001 Bt PS80JJl

Cry35Aal

Crv34Aa2 AAK64560 Rupar et al Binary with

2001 Bt EG5899

Cry35Aa2

Cry34Aa3 AAT29032 Binary with

Schnepf et al 2004 Bt PS69Q

Cry35Aa3

Crv34Aa4 AAT29030 Binary with

Schnepf et al 2004 Bt PS185GG

Cry35Aa4 Binary with

Crv34Abl AAG41671 Moellenbeck et al 2001 Bt PS 149B l

Cry35Abl Binary with

Cry34Acl AAG501 18 Ellis et al 2001 Bt PS167H2

Cry35Acl

Crv34Ac2 AAK64562 Rupar et al Binary with

2001 Bt EG9444

Cry35Ab2 Binary with

Crv34Ac3 AAT29029 Schnepf et al 2004 Bt KR1369

Cry35Ab3

Cry34Bal AAK64565 Rupar et al Binary with

2001 Bt EG4851

Cry35Bal Crv34Ba2 AAT29033 Schnepf et al 2004 Bt PS201L3 Binary with Cry35Ba2

Crv34Ba3 AAT29031 Schnepf et al Binaiy with

2004 Bt PS201HH2

Cry35Ba3

Crv35Aa1 AAG50342 Ellis et al Binary with

2001 Bt PS80JJl

Cry34Aal

Cry35Aa2 AAK64561 upar et al Binary with

2001 Bt EG5899

Cry34Aa2

Crv35Aa3 AAT29028 Schnepf et al Binary with

2004 Bt PS69Q

Cry34Aa3

Cry35Aa4 AAT29025 Schnepf et al Binary with

2004 Bt PS185GG

Cry34Aa4

Crv35Abl AAG41672 Binary with

Moellenbeck et al 2001 Bt PS 149B l

Cry34Abl

Crv35Ab2 AAK64563 Rupar et al Binary with

2001 Bt EG9444

Cry34Ac2

Crv35Ab3 AY536891 Binaiy with

AAT29024 2004 Bt KR1369

Cry34Ab3

Crv35Ac l AAG501 17 Ellis et al Binary with

2001 Bt PS167H2

Cry34Acl

Crv35B l AAK64566 Rupar et al Binary with

2001 Bt EG4851

Cry34Bal

Cry35Ba2 AAT29027 Schnepf et al Binary with

2004 Bt PS201L3

Cry34Ba2

Crv35Ba3 AAT29026 Schnepf et al Binary with

2004 Bt PS201HH2

Cry34Ba3

Crv36Aal AAK64558 Rupar et al 2001 Bt

Cry37Aa l AAF76376 Donovan et al Binary with

2000 Bt

Cry23Aa

Cry38Aa] AAK64559 Rupar et al 2000 Bt

Crv39Aal BAB72016 Ito et al 2001 Bt aizawai

Crv40Aa1 BAB72018 Ito et al 2001 Bt aizawai

Crv40Ba l BAC77648 Ito et al 2003 Bunl-14

Cry40Cal EU381045 Shu et al No NCBI link

2008 Bt Y41

July09

Crv40Dal ACF15199 Zhang et al 2008 Bt S2096-2

Crv41Aal BAD35157 Yamashita et al 2003 Bt A 1462

Crv41Abl BAD35163 Yamashita et al 2003 Bt A1462

Crv42Aal BAD35166 Yamashita et al 2003 Bt A1462

Yokoyama and

Cry43Aal BAD15301 P. lentimorbus

2003

Tanaka semadara

Crv43Aa2 BAD95474 P. popilliae

Nozawa 2004

popilliae

Yokoyama and

Cry43Bal BAD 15303 P. lentimorbus

2003

Tanaka semadara

Yokoyama and

Crv43-like BAD 15305 P. lentimorbus

2003

Tanaka semadara Bt entomocidus

Crv44Aa BAD08532 Ito et al 2004

ΓΝΑ288

Crv45Aa BAD22577 Okumura et al 2004 Bt 89-T-34-22

Crv46Aa BAC79010 Ito et al 2004 Bt dakota

Crv46Aa2 BAG68906 Ishikawa et al 2008 Bt A 1470

Crv46Ab BAD35170 Yamagiwa et al 2004 Bt

Crv47Aa AAY24695 Kongsuwan et al 2005 Bt CAA890

Crv48Aa CAJ18351 Jones and Berry 2005 Bs IAB59 binary with 49 Aa binary with

Crv48Aa2 CAJ86545 Jones and Berry 2006 Bs 47-6B

49Aa2 binaiy with

Crv48Aa3 CAJ86546 Jones and Berry 2006 Bs NHA15b

49Aa3 binary with

Crv48Ab CAJ86548 Jones and Berry 2006 Bs LP 1G

49AM binaiy with

Crv48Ab2 CAJ86549 Jones and Berry 2006 Bs 2173

49Aa4

Crv49Aa CAH56541 Jones and Berry 2005 Bs IAB59 binary with 48Aa binaiy with

Crv49Aa2 CAJ86541 Jones and Berry 2006 Bs 47-6B

48Aa2 binary with

Crv49Aa3 CAJ86543 Jones and Berry 2006 BsNHA15b

48Aa3 binaiy with

Crv49Aa4 CAJ86544 Jones and Berry 2006 Bs 2173

48Ab2 binary with

Crv49Abl CAJ86542 Jones and Berry 2006 Bs LP 1G

48AM

Crv50Aal BAE86999 Ohgushi et al 2006 Bt sotto

CryS lAal ABI 14444 Meng et al 2006 Bt F14-l

Cry52Aal EF613489 No NCBI link

Song et al 2007 Bt Y41

July09

Cry52Bal FJ361760 No NCBI link

Jun et al 2008 Bt BM59-2

July09

Cry53Aal EF633476 Song et al No NCBI link

2007 Bt Y41

July09

No NCBI link

Cry53Abl FJ361759 Jun et al 2008 Bt MC28

July09

Crv54Aal ACA52194 Tan et al 2009 Bt MC28

Crv55Aa1 ABW8893 1 Guo et al 2008 YBT 1518

Crv55Aa2 AAE33526 Bradfisch et al 2000 BT Y41

Cry56Aal FJ597621 No NCBI link

Jun & Furong 2008 Bt Ywc2-8

July09

Cry56Aa2 GQ483512 No NCBI link

Guan Peng et al 2009 Bt G7-1

Aug09

Crv57Aal ANC87261 Noguera & Ibarra 2009 Bt kim

Crv58Aa1 ANC87260 Noguera & Ibarra 2009 Bt entomocidus

Crv59Aal ACR43758 Noguera & Ibarra 2009 Bt kim LBIT-98(

Claims

We claim:

1. A transgenic plant comprising DNA encoding a Vip3 Ab insecticidal protein and DNA encoding a CrylCa insecticidal protein.

2. The transgenic plant of claim 1, said plant further comprising DNA encoding a third insecticidal protein, said third protein being selected from the group consisting of Cry 1 Fa, Cry 1 Da, Cry 1 Be, and Cry IE.

3. The transgenic plant of claim 2, wherein said third protein is selected from the group consisting of CrylFa and CrylBe, said plant further comprising DNA encoding fourth and fifth insecticidal proteins selected from the group consisting of Cry2A, Cry II, DIG-3, and CrylAb.

4. Seed of a plant according to any of claims 1-3, wherein said seed comprises said DNA.

5. A field of plants comprising non-Bt refuge plants and a plurality of plants according to any of claims 1-3, wherein said refuge plants comprise less than 40% of all crop plants in said field.

6. The field of plants of claim 5, wherein said refuge plants comprise less than 30% of all the crop plants in said field.

7. The field of plants of claim 5, wherein said refuge plants comprise less than 20% of all the crop plants in said field.

8. The field of plants of claim 5, wherein said refuge plants comprise less than 10% of all the crop plants in said field.

9. The field of plants of claim 5, wherein said refuge plants comprise less than 5% of all the crop plants in said field.

10. The field of plants of claim 5, wherein said refuge plants are in blocks or strips.

1 1. A mixture of seeds comprising refuge seeds from non-Bt refuge plants, and a

plurality of seeds of claim 4, wherein said refuge seeds comprise less than 40% of all the seeds in the mixture.

12. The mixture of seeds of claim 11, wherein said refuge seeds comprise less than 30% of all the seeds in the mixture.

13. The mixture of seeds of claim 11, wherein said refuge seeds comprise less than 20% of all the seeds in the mixture.

14. The mixture of seeds of claim 11, wherein said refuge seeds comprise less than 10% of all the seeds in the mixture.

15. The mixture of seeds of claim 11, wherein said refuge seeds comprise less than 5% of all the seeds in the mixture.

16. A method of managing development of resistance to a Cry protein by an insect, said method comprising planting seeds to produce a field of plants of claim 5.

17. A field of any of claims 5-10, wherein said plants occupy more than 10 acres.

18. A plant of any of claims 1-3, wherein said plant is selected from the group

consisting of corn, soybeans, and cotton.

19. The plant of claim 18, wherein said plant is a maize plant.

20. The transgenic plant of claim 1, said plant further comprising DNA encoding a CrylFa insecticidal protein.

21. A plant cell of a plant of any of claims 1 -3 , wherein said plant cell comprises said DNA encoding said Vip3Ab insecticidal protein and said DNA encoding said CrylCa insecticidal protein, wherein said Vip3Ab insecticidal protein is at least 99% identical with SEQ ID NO: l, and said CrylCa insecticidal protein is at least 99% identical with SEQ ID NO:2.

22. A plant of any of claims 1-3, wherein said Vip3Ab insecticidal protein comprises SEQ ID NO: l, and said CrylCa insecticidal protein comprises SEQ ID NO:2.

23. A method of producing the plant cell of claim 21.

24. A method of controlling a fall armyworm insect by contacting said insect with a Vip3Ab insecticidal protein and a CrylCa insecticidal protein.